Product and method for removal of biofilms

ABSTRACT

A method for removing a biofilm present on a substrate includes providing a detergent component comprising a sequestrant, a wetting agent and a dispersant, and an enzymatic component containing at least one protease, and at least one polysaccharidase in solution in an aqueous phase in sequence or simultaneously, and applying the solution to the substrate for a predetermined period of time to effect biofilm removal from the substrate.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/044,309, filed Jul. 24, 2018, titled PRODUCT AND METHOD FOR REMOVALOF BIOFILMS, which is a divisional of U.S. application Ser. No.14/234,534, filed Apr. 21, 2014, now abandoned, titled PRODUCT ANDMETHOD FOR THE REMOVAL OF BIOFILMS, which is a National Stage ofInternational Application No. PCT/EP2010/065567, filed Oct. 15, 2010,titled PRODUCT AND METHOD FOR THE REMOVAL OF BIOFILMS, the disclosuresof which are hereby incorporated by reference in their entirety.

BACKGROUND

The invention relates to the field of biofilm removal. Morespecifically, the invention pertains to a composition and a method forremoving biofilms.

Hygiene is of increasing importance in the food industry, in hospitalsand particularly for surgery, in water purifying and desalinatingplants, in the treatment of process water and in particular waste waterof cooling towers, and for everyday needs such as contact lenses. Whenwater circulates on a substrate, it is frequently observed thatmicroorganisms freely circulating in the water may adhere to thesurface. These microorganisms may then develop an adhesive extracellularmatrix composed of polymer substances. A community of microorganismsadhering to a surface and encompassed in said matrix is called abiofilm. In general these biofilms are composed of bacteria.

It is unfortunately observed that this matrix is highly resistant andmay form a barrier for agents acting against the microorganisms.Conventional treatments using sodium hydroxide and/or comprisingdifferent biocides do not act in sufficiently efficient manner sincethey do not penetrate the biofilm over its entire thickness or areinhibited by some constituent molecules of this matrix. Treatment isthen only partly effective on the top surface of the biofilm. Inaddition, this film may also trap other microorganisms, particularlypathogenic microorganisms, than those initially present.

From WO09/26807 an enzymatic method is known for treating a biologicalfilm. In this method the biofilm is placed in contact with a cleaningcomposition containing one or more hydrolases to remove or release thebiofilm layer from the surface. At a second step the biofilm iscontacted with a bactericide disinfecting composition to destroy thebacterial cells present in the film. However, the simultaneous use ofthese two compositions is the cause of a degree of inactivation of someenzymes in the final mixture. The rapidity and efficacy of cleaning cantherefore be improved by using a composition in which there is noinactivation of enzymes.

From document US2003/0205247 the use is also known of aqueous solutionscontaining enzymes to clean storage or fermentation tanks comprising oneor more enzymes chosen from among the following: laccases, peroxidases,oxidoreductases, transferases, isomerases, lyases or ligases and athickening agent with a foam amplifier. The field of action of theaforementioned solutions is relatively narrow, suitable for breweriessince the chosen, illustrated enzymes are particularly known to beactive on polyphenols which are the main constituents of fermentationresidues, tannins and similar. The thickening agent is an agentmodifying the viscosity and thixotropy of the solution, and is used forthe purpose of allowing better adherence of the solution and/or of thefoam onto the surfaces of the tanks to be treated. These solutions arenot suitable for cleaning other tank or reservoir installations (e.g.comprising multiple piping or tubing) and are not suitable for removingother types of microorganisms and hence for a broad range of biofilms.

Document WO 92/13807 discloses the use of a composition allowing theremoval of biomass and biofilms on substrates in aqueous systems i.e. insystems in which water is set in circulation or deposited. This type ofsystem suffers from the presence of alkaline or acid biomass andbiofilms due to the type of organisms generally present in this type ofplant.

To solve this problem, document WO 92/13807 provides for the use ofpolysaccharides and/or proteases and anionic surfactants such as SOS orDBS (sodium dodecylsulphate and dodecylbenzenesulphonic acidrespectively) to remove biomass and biofilms.

Unfortunately, this type of composition is not efficient for a widerange of biofilms caused by various microorganisms and is of limitedefficacy in removing biofilms.

As can be ascertained from the foregoing, these compositions alwaystarget a type of biofilm or particular microorganism and/or a particularapplication.

There is therefore a need for a composition and method capable ofremoving biofilms which are efficient within a reasonable time period,act on a wide class of biofilms produced by a wide class ofmicroorganisms or groups of microorganisms, which are not harmful forthe biofilm substrate and act both to prevent the development ofbiofilms and on longstanding biofilms having reached a stage of cohesionand major resistance.

To solve this problem, the present invention provides a composition forthe removal of biofilms present on a substrate characterized in that itcomprises:

-   -   at least one detergent component comprising a sequestrant, a        wetting agent and a dispersant,    -   at least one enzymatic component containing at least one        protease, at least one laccase and at least one        polysaccharidase.

The forming of a composition comprising a detergent component and anenzymatic component containing at least one protease, at least onelaccase and at least one poly saccharidase surprisingly allows asignificant improvement in the rapidity and efficacy of the removal of abiofilm whilst being able to attack various types of biofilms. Thiscomposition allows the removal of all or almost all of the biofilm andcan act on biofilms that are even mature or having an early developmentcycle and developed by multiple different species or microorganisms;

To date it is to be pointed out that no efficient composition can ensurethe complete removal of biofilms present industrial plants. For example,in the food industry biofilms are inevitably formed (having regard tothe richness of the surrounding medium). Biofilms have cyclic growthactivity comprising a growth phase during which the microorganismsaccumulate and a detachment phase during which entire pieces of biofilmsbecome detached by erosion and under the effect of their own weight.When an industrialist is faced with this phenomenon, the production lineshould be stopped and different alternate washing cycles carried outwith sodium hydroxide and numerous detergents and/or chemical and/orenzymatic cleaning agents since no polyvalent composition is available.Yet this represents numerous hours of work and loss of production yield.

As a result, in practice, production is not stopped and when the biofilmreaches the rupture phase the batches produced which are contaminatedare discarded until the level of microorganism contamination of the foodproduced drops to a level acceptable by standards in force. In addition,it cannot be envisaged by industrialists to stock a particular detergentor enzymatic solution for every microorganism likely to contaminate theproduction line.

It is therefore most surprisingly that the present invention allows theproviding of a fully polyvalent detergent composition which allows theremoval of a broad spectrum of biofilms in various types ofinstallations, does not require any particular precautions of use and isapplicable both to tank type installations and to pipe typeinstallations. The detergent removes a surface part of the biofilm andwets and/or swells the organic structures of the biofilm through thedispersing nature of the dispersant present in the detergent component.This therefore promotes the accessibility of the enzymatic componentwhich weakens and degrades the matrix of the biofilm. This combinedaction of the three types of enzyme and of the detergent componentpromotes the accessibility of the composition to the deeper layers andallows optimal detaching of any type of biofilm whilst preserving thesubstrate.

In one particular embodiment of the invention, the composition is asolution with a pH of approximately between 8 and 11, preferably betweenapproximately 9.5 and 10.5 and more preferably between approximately 9.5and 10.

The pH value of the said composition has a major influence on itsefficacy against a biofilm. A solution of said composition whose pH isapproximately between 8 and 11 therefore surprisingly allows the removalof all or almost all of a biofilm.

Alternatively, said composition can be in solid form that is dissolvedbefore use in a solvent so that when it is diluted in an aqueous phase,before application onto a biofilm, a solution is obtained whose pH isapproximately between 8 and 11. In this case, the solid composition maycomprise a basic compound and when it is placed in solution in thesolvent and then diluted in an aqueous phase before application to thebiofilm, a buffered solution is obtained having the composition of theinvention with alkaline pH (value between 8 and 11).

In one advantageous variant of the invention, the composition is asolution designed so that when it is diluted in an aqueous phase beforeapplication to a biofilm, it forms a solution having a pH ofapproximately between 6.5 and 7.5, more particularly of about 7. In thismanner, the pH of the solution of the composition is particularlysuitable for the action of the enzymatic component, of laccase inparticular. In addition, provision is made according to the invention toincrease the pH at a subsequent application phase of the composition ofthe invention so as to obtain an alkaline pH particularly suitable forremoving biofilm as mentioned above whilst maintaining optimum efficacyof the enzymatic component.

Alternatively, the said composition can be in solid form which isdissolved before use in a solvent to obtain a solution which is laterdiluted in an aqueous phase to obtain a cleaning solution having a pH ofbetween approximately 6.5 and 7.5, preferably about 7.

Preferably, the said at least one enzymatic component comprises aproportion of protease(s) of between 10 and 50%, a proportion oflaccase(s) of between 5 and 35% and a proportion of polysaccharidase(s)of between 5 and 20% by weight relative to the total weight of theenzymatic composition, the 100% of the enzymatic component optionallybeing reached using a conventional excipient or solvent e.g. an alcohol.

According to one preferred embodiment of the invention, the enzymaticcomponent may contain between 1 and 10 proteases, preferably between 1and 5 proteases, more preferably it may contain 2, 3, 4 or 5 proteases.

Non-limiting examples of protease enzymes belonging to class EC 3.4 andable to be used in the invention are the amino-peptidases (EC 3.4.11),dipeptidases (EC 3.4.13), dipeptidyl-peptidases andtripeptidyl-peptidases (EC 3.4.14), peptidyl-dipeptidases (EC 3.4.15),serine carboxypeptidases (EC 3.4.16), metallo carboxypeptidases (EC3.4.17), cysteine carboxypeptidases (EC 3.4.18), omega peptidases (EC3.4.19), serine endopeptidases (EC 3.4.21), cysteine endopeptidases (EC3.4.22), aspartic endopeptidases (EC 3.4.23), metallo endopeptidases (EC3.4.24), threonine endopeptidases (EC 3.4.25) and endopeptidasesbelonging to class EC 3.4.99.

Preferably, the proteases belong to class EC 3.4.21. The proteases arecommercially available and are in different forms including powders,granules, suspensions, liquid solutions.

The laccases used in the invention belong to class EC 1.10.3.2. Laccasesare enzymes containing copper and have the role of oxidizing a substratein the presence of oxygen. More specifically, laccases areoxidoreductases which act with molecular oxygen as electron acceptor.

The at least one polysaccharidase used in the invention is an enzymewhose function is to break up the bonds in polysaccharides. Preferably,the at least one polysaccharidase may be an alpha-amylase, cellulose,hemi-cellulase, glucosidase, beta-glucanase or pectinase.

More preferably, the at least one polysaccharidase may be analpha-amylase belonging to class EC 3.2.1.1, whose function is to breakup (1-4)-alpha-glycosidic bonds in polysaccharides containing three ormore alpha-(1-4)-D-glucose units. Preferably, the enzymatic componentmay comprise a proportion of laccase(s) of approximately 30%, aproportion of protease(s) of approximately 30%, a proportion ofalpha-amylase(s) of approximately 10% by weight relative to the totalweight of the enzymatic component, the 100% of the enzymatic componentoptionally being reached using a conventional excipient or solvent.

According to another preferred embodiment, if the enzymatic componentcomprises 2 proteases, the proportion of laccaseos may be approximately30%, the total proportion of proteases approximately 30%, the proportionof alpha-amylase(s) approximately 10% by weight relative to the totalweight of the enzymatic component, the 100% of enzymatic componentoptionally being reached using a conventional excipient or solvent.

For example, the ratio between each protease may be between 1:2 and 2:1,preferably the ratio between each protease may be 1:1. The enzymespresent in the enzymatic component have complementary action on abiofilm. For example, laccase is highly effective against contaminationnot attacked by alpha-amylase or proteases.

According to one preferred embodiment of the invention, the enzymaticcomponent may be a solution or in solid form.

Preferably, the enzymatic component is a solution whose pH is betweenapproximately 8 and 10. Preferably the enzymatic component is an aqueoussolution whose pH may be between approximately 8.5 and 9.5; morepreferably the pH may be approximately 9.0 for maximum maintaining ofenzyme integrity.

Alternatively, the enzymatic component may be in solid form such as alyophilisate, a powder, granules or in any other form allowing thesolubilizing of said component in a solvent, which is later dissolved insaid solvent. The solvent may be water or an aqueous, acid, basic,alcohol, buffered or neutral solution. The solubilized enzymaticcomponent in this case can then subsequently be diluted in an aqueoussolution optionally containing one or more compounds such as detergentsfor example to form the cleaning solution.

In one advantageous embodiment of the invention, the said at least onedetergent component comprises a proportion of sequestrant of between 1and 10% by weight relative to the total weight of the detergentcomponent, which represents an optimum between efficacy, stability andcost.

The sequestrant is a chemical substance capable of forming complexeswith mineral ions which it fixes in a form preventing the precipitationthereof via usual reactions. For example, the sequestrant may beethylene-diamine-tetraacetic acid, glucono-delta-lactone, sodiumgluconate, potassium gluconate, calcium gluconate, citric acid,phosphoric acid, tartaric acid, sodium acetate, sorbitol, a compoundcontaining a phosphorus atom. Preferably, the sequestrant may be aphosphorus oxide such as a phosphonate, phosphinate or phosphate or amixture thereof, or a salt thereof, an amine or amine oxide carrying inits structure at least one functional phosphine, phosphine oxide,phosphinite, phosphonite, phosphite, phosphinate, phosphinate orphosphate group, alone or in combination or a salt thereof.

More preferably the sequestrant may be a phosphonate or a salt thereof,an amine or amine oxide containing in its structure at least onefunctional phosphine, phosphine oxide, phosphinite, phosphonite,phosphite, phosphonate, phosphinate or phosphate group, alone or incombination, or a salt thereof. As a non-limiting example, thephosphonate may have the general formula R¹(R²—O)(R³O)P═O in which R¹,R² and R³ independently represent a hydrogen, alkyl, substituted alkyl,substituted or non-substituted alkyl-amino, substituted ornon-substituted aminoalkyl, aryl or substituted aryl group. As anon-limiting example, the amine or amine oxide may comprise one, two orthree substituents of general formula CR⁴R⁵W in which R⁴ and R⁵independently of each other represent a hydrogen, alkyl, substitutedalkyl, substituted or non-substituted alkyl-amino, substituted ornon-substituted aminoalkyl, aryl or substituted aryl group, and W is aphosphonate, phosphinate or phosphate group. The sequestrant may be inthe form of a sodium, calcium, lithium, magnesium or potassium salt;preferably the sequestrant may be in the form of a sodium, calcium orpotassium salt.

In one advantageous variant of the invention, the detergent componentcomprises a proportion of dispersant of between 1 and 10% by weightrelative to the total weight of the detergent component.

The dispersant is therefore a chemical substance capable of improvingthe separation of the particles of a suspension to preventagglutination, aggregation and/or decanting. The dispersant may be apolymer soluble or partly soluble in water such as polyethylene glycolfor example or cellulose derivatives or a polymer comprising at leastone acrylic acid or acrylic ester repeat unit. Preferably, thedispersant is a polymer comprising at least one acrylic acid or acrylicester repeat unit of general formula —(CH₂—CH—COOR)— in which Rrepresents a hydrogen, alkyl or substituted alkyl, aryl or substitutedaryl group. In particular, the dispersant is a polymer having a weightaverage molecular weight Mw of approximately between 500 and 10000.

More preferably, the dispersant is a polymer of acrylic acid. Inparticular, the dispersant may be a homopolymer of acrylic acid having aweight average molecular weight of approximately between 2000 and 6000.

In another embodiment of the invention, the detergent componentcomprises a proportion of wetting agent of between 1 and 15% by weightrelative to the total weight of the detergent component.

The wetting agent is an amphiphilic chemical substance or a compositioncomprising said amphiphilic chemical substance, which modifies thesurface tension between two surfaces. The advantage of the wetting agentis to promote the spreading of a liquid on a solid. The wetting agentmay be anionic, cationic, non-ionic or zwitterionic. Preferably, thewetting agent may be an anionic or non-ionic wetting agent i.e. thehydrophilic part is negatively charged or does not comprise any distinctcharge or may be a composition comprising an anionic wetting agent. Moreparticularly, the wetting agent may be a sucrose ester or a compositioncomprising a sodium alkyl sulphate and an alcohol.

Advantageously, and preferably, in the detergent component of theinvention, the said wetting agent is non-foaming when hot and ispreferably chosen from the group formed by C₆ to C₁₀ sodium alkylsulphates, C₆ to C₁₀ alcohol ether sulphates, C₆ to C₁₀ alkyl arylsulphonates.

The fact that the wetting agent is a non-foaming wetting agent when hotallows the use thereof in installations comprising numerous pipes andtubes, thereby preventing the formation of foam without deterioratingthe surfactant and/or emulsifying performance of the compositionaccording to the invention, indeed the contrary. Evidently, theproviding of an efficient detergent solution which does not generatefoam limits the rinsing steps, which is particularly desirable inparticular in installations having multiple piping and tubing.

As for the enzymatic component, the detergent component may be in theform of a solid to be dissolved in a solvent and/or in an aqueous phaseor in liquid form.

When in solid form, it can either be directly placed in solution in thesolution formed by the enzymatic component optionally already diluted inthe aqueous phase, or it can be placed in solution in a solvent, priorto its dilution in the solution formed by the enzymatic component andthe aqueous phase, or in the aqueous phase directly before dilution ofthe enzymatic component.

If the detergent component is in liquid form, the 100% of the detergentcomponent are in general optionally reached using water, and prior toapplication to the biofilm it is diluted in an aqueous phase optionallyalready containing the enzymatic component.

Other forms of embodiment of the composition according to the inventionare mentioned in the appended claims.

A further subject of the invention is a method for removing biofilmspresent on a substrate, characterized in that it comprises the followingsteps of:

-   -   a) providing a detergent component comprising a sequestrant,        wetting agent and dispersant, and an enzymatic component        containing at least one protease, at least one laccase and at        least one polysaccharidase;    -   b) placing the detergent component in solution in an aqueous        phase;    -   c) placing the enzymatic component in solution in the solution        formed at step b) to form the solution of said composition        according to the invention; or:    -   b′) placing the enzymatic component in solution in an aqueous        phase;    -   c′) placing the detergent component in solution in the solution        formed at step b′) to form the solution of said composition        according to the invention;    -   d) applying the solution of said composition formed at step c)        or c′) to the substrate for a predetermined period of time, in        particular of between 15 minutes and 4 hours.

By “placing in solution” in the present invention is meant either theplacing in solution of a solid compound in a liquid phase (solvent) orthe dilution of a liquid compound in another liquid phase with which theliquid compound is miscible.

Alternatively, steps b) and c), or b′) and c′) can be conductedsimultaneously to form a solution of said composition of the invention.

Preferably the method comprises the following steps of:

-   -   a) providing a detergent component containing a sequestrant, a        wetting agent and a dispersant; and an enzymatic component        containing at least one protease, at least one laccase and at        least one polysaccharidase;    -   b) placing the detergent component in solution in water;    -   c) placing the enzymatic component in solution in the solution        formed at step b) to form the solution of said composition of        the invention;    -   d) applying the solution of said composition formed at step c)        to the substrate for a predetermined period of time, preferably        of between 15 minutes and 4 hours.

According to one preferred embodiment of the invention, the pH of thesolution formed at step b) is between approximately 11.0 and 14.0,preferably between approximately 12.0 and 14.0 and more preferablybetween 12.8 and 13.8.

Advantageously, the pH of the solution of said composition formed atstep c) or c′) is between approximately 8 and 11, preferably betweenapproximately 9.5 and 10.5, and more preferably between approximately9.5 and 10.

In one variant of the invention, the pH of the solution of saidcomposition formed at step c) or c′) is between approximately 6.5 and7.5, and a basic solution is added after the said application d) of thesolution of said composition to the said substrate during saidpredetermined period of time so as to increase the pH up to about 8 to9.

In this manner, when the composition of the invention is applied to thesubstrate, the prevailing pH is approximately between 6.5 and 7.5enabling the laccase to benefit from optimal activity conditions. Next,the fact that the pH is increased up to about 8 or 9 enables the otherenzymes in turn to reach their optimal efficacy. In this manner, theytoo will reach optimal activity conditions and as a result the biofilmwill be removed in particularly advantageous manner since each enzymepresent will find optimal conditions to perform its action and thebiofilm will be detached and fully removed. In addition, since basiccomponents are generally used in installations in which a biofilm mayform, there is no addition of any exogenous component which could raiseproblems for validating the cleansing step. For example, to sanitize aninstallation, sodium hydroxide is frequently used and hence the additionof a substance of different nature is limited in the present invention.

Preferably the temperature of the solution of the detergent componentformed at step b) or c′) may be between approximately 35° C. and 50° C.According to one preferred embodiment of the invention, the compositionof the invention is applied to a substrate coated with a biofilm forapproximately 30 to 50 minutes, which represents a relatively shortapplication time for such efficacy.

Preferably, the detergent compound comprises a proportion of sequestrantof between 1 and 10%, a proportion of dispersant of between 1 and 10%,and a proportion of wetting agent of between 1 and 15% by weightrelative to the total weight of the detergent component. Preferably, theenzymatic component comprises a proportion of protease(s) of between 10and 50%, a proportion of laccase(s) of between 5 and 35% and aproportion of polysaccharidase(s) of between 5 and 20% by weightrelative to the total weight of the enzymatic component. Morepreferably, the at least one polysaccharidase can be an alpha-amylase.

With the present method, it is possible efficiently to remove theentirety of near-entirety of the biofilm, leaving on the substrate onlyisolated cells without the protection of the matrix. The subsequentaction of a biocide allows the microbial strain to be destroyed. Asubsequent disinfection phase will therefore be much more efficientafter the application of a solution of the composition of the inventionthan after the application of a conventional cleaning phase not allowingthis full removal of the matrix.

Therefore according to one preferred embodiment of the invention, themethod further comprises a subsequent step to apply a biocide. Forexample but not limited thereto, the biocides may be of oxidizing typesuch as peracetic acid, hydrogen peroxide, potassium monopersulfate,sodium hypochlorite. According to the invention, the application of abiocide must be made after the application of the composition of theinvention to prevent deactivation of the enzymes present in the saidcomposition by the biocides.

Other embodiments of the method of the invention are mentioned in theappended claims.

The invention also relates to the use of a composition of the inventionto remove biofilms present on a substrate, in particular to clean floorsand surfaces, for clean-in-place or immersion cleaning. The compositioncan be used in closed installations or for immersion. Cleaning byimmersion is particularly used to clean surgical equipment, contactlenses. The composition of the invention can be used to clean technicaland process water circuits, air conditioning exchange systems or in thefood industry.

Other forms of use according to the invention are mentioned in theappended claims.

The present invention also relates to a kit for the removal of biofilmspresent on a substrate, characterized in that it comprises:

-   -   at least one sample of detergent component in solution or in        solid form, containing a sequestrant, a wetting agent and a        dispersant; and    -   at least one sample of enzymatic component in solution or in        solid form containing at least one protease, at least one        protease, at least one laccase and at least one        polysaccharidase.

Preferably the at least one polysaccharidase may be an alpha-amylase.

According to one preferred embodiment of the invention, the sample ofthe enzymatic component may contain between 1 and 10 proteases,preferably between 1 and 5 proteases, more preferably it may contain 2,3, 4 or 5 thereof.

According to another preferred embodiment, the sample of the enzymaticcomponent contained in the kit may comprise 2 proteases. Preferably, ifthe sample of enzymatic component comprises 2 proteases, the proportionof laccase(s) may be approximately 30%, the total proportion ofproteases may be approximately 30% and the proportion ofalpha-amylase(s) may be approximately 10% by weight relative to thetotal weight of the enzymatic component.

For example, the ratio between each protease may be between 1:2 and 2:1,preferably the ratio between each protease may be 1:1.

Preferably, the sample of the detergent component comprises a proportionof sequestering agent of between approximately 1 and 10%, a proportionof dispersant of between approximately 1 and 10% and a proportion ofwetting agent between approximately 1 and 15% by weight relative to thetotal weight of the detergent component, optionally the 100% of thedetergent component possibly being reached using water.

As an example, the sequestrant may be ethylene-diamine-tetraacetic acid,glucono-delta-lactone, sodium gluconate, potassium gluconate, calciumgluconate, citric acid, phosphoric acid, tartaric acid, sodium acetate,sorbitol, a compound containing a phosphorus atom. Preferably, thesequestrant can be a phosphorus oxide such as phosphonate, phosphinateor phosphate or a salt thereof, an amine or amine oxide or a saltthereof carrying in its structure at least one functional phosphine,phosphine oxide, phosphinite, phosphonite, phosphite, phosphonate,phosphinate or phosphate group.

More preferably the sequestrant may be a phosphonate or a salt thereof,an amine or amine oxide, or salt thereof, comprising in its structure atleast one functional phosphine, phosphine oxide, phosphinite,phosphonite, phosphite, phosphonate, phosphinate or phosphate group. Asa non-limiting example, the phosphonate may have the general formulaR¹(R²O)(R³O)P═O in which R¹, R² and R³ are independently selected fromthe hydrogen, alkyl, substituted alkyl, substituted or non-substitutedalkyl-amino, substituted or non-substituted aminoalkyl, aryl orsubstituted aryl groups. As a non-limiting example, the amine or amineoxide may comprise one, two or three substituents of general formulaCR⁴R⁵W in which R⁴ and R⁵ are independently selected from the hydrogen,alkyl, substituted alkyl, substituted or non-substituted alkyl-amino,substituted or non-substituted aminoalkyl, aryl or substituted arylgroups, and W is selected from the phosphonate, phosphinate or phosphategroup. The sequestrant may be in the form of sodium, calcium, lithium,magnesium or potassium salt; preferably the sequestrant may be in theform of a sodium, calcium or potassium salt.

The dispersant may be a water-soluble or partly water-soluble polymersuch as polyethylene glycol, cellulose derivatives or a polymercomprising at least one acrylic acid or acrylic ester repeat unit.Preferably, the dispersant is a polymer comprising at least one acrylicacid or acrylic ester repeat unit of general formula —(CH₂—CH—COOR)— inwhich R may be a hydrogen, alkyl or substituted alkyl, an aryl orsubstituted aryl. In particular, the dispersant is a polymer having aweight average molecular weight Mw of between approximately 500 and10000.

More preferably, the dispersant is a polymer of acrylic acid. Inparticular, the dispersant may be a homopolymer of acrylic acid having amolecular weight of between approximately 2000 and 6000.

The wetting agent may be anionic, cationic, non-ionic or zwitterionic.Preferably, the wetting agent may be an anionic or non-ionic wettingagent i.e. the hydrophilic part is negatively charged or has no distinctcharge or it may be a composition comprising an anionic wetting agent

More particularly, the wetting agent may be a composition comprising asodium alkyl sulphate and an alcohol.

Advantageously, in the kit of the invention, the sample of the enzymaticcomponent is an aqueous solution with a pH approximately between 8 and10, preferably between approximately 8.5 and 9.5 and more preferably ofabout 9.0.

If the sample of the detergent component is a solution, the pH thereofis approximately between 11.0 and 14.0, more preferably betweenapproximately 12.0 and 14.0, most preferably between 12.8 and 13.8.

In one variant of the invention, the sample of the enzymatic componentis an aqueous solution whose pH is between approximately 6.5 and 7.5,preferably about 7.

Preferably, the kit of the invention also comprises a biocide.

Other embodiments of the kit according to the invention are mentioned inthe appended claims.

Other characteristics, details and advantages of the invention willbecome apparent from the description below given as a non-limitingexample with reference to the appended drawings and to the Examples.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a graph showing the cell quantity of a Pseudomonas fluorescensstrain on a substrate after application of different treatmentsolutions.

FIG. 2 is a graph showing the cell quantity of a Bacillus mycoidesstrain on a substrate after application of different treatmentsolutions.

FIG. 3 is a graph showing the cell quantity of a Bacillus cereus strainon a substrate after application of different treatment solutions.

FIG. 4 gives images of the surface of a substrate, taken underepifluorescence microscopy, after application of different treatmentsolutions to said substrate coated with a biofilm of the strainPseudomonas fluorescens.

FIG. 5 gives images of the surface of a substrate taken underepifluorescence microscopy after application of different treatmentsolutions to said substrate coated with a biofilm of the strain Bacillusmycoides.

FIG. 6 gives images of the surface of a substrate taken underepifluorescence microscopy after application of different treatmentsolutions to said substrate coated with a biofilm of the strain Bacilluscereus.

FIG. 7 gives the results of staining of biofilms before cleaning withcleaning solutions (A) and after cleaning with said solutions (B).

FIG. 8 is a graph showing the efficacy of four enzymes against biofilmsderived from the strain Chryseobacterium meningosepticum.

FIG. 9 is a graph showing the efficacy of four enzymes against biofilmsderived from the strain Bacillus Cereus.

FIG. 10 is a graph showing the efficacy of four enzymes against biofilmsderived from the strain Pseudomonas fluorescens.

DETAILED DESCRIPTION

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

In the Figures, identical or similar elements carry the same references.

Example 1

Description of chosen test conditions: the tests were conducted as partof Clean-In-Place (CIP) procedure in a pilot industrial plant. Thebiofilms were developed on a planar face of a stainless steel cylinderperforated in its centre. This cylinder, once placed in a pipe havingthe same inner diameter as the outer diameter of the cylinder, causessudden narrowing of the diameter thereof forcing liquid to pass throughthe central perforation. This stress leads to flow perturbations therebycreating dead zones on the surface of the cylinder. These dead zonespromote the formation of a biofilm and hinder its mechanical detachmentby the flow. They therefore typically represent zones that are difficultfor biofilm removal.

Three different strains were used: Pseudomonas fluorescens (supplied byUniversity of Cornell, Department of Food Science, Ithaca, N.Y., 14853,USA (Kathryn J. Boor, kjb4@cormell.edu); Bacilfus mycoides (supplied bythe AFSSA laboratory in Maison-Alfort, France (Brigitte Carpentier,b.carpentier@lerpac.afssa.fr); Bacillus cereus (supplied by INRA, UR638,Processus aux Interfaces et Hygiene des Materiaux, Villeneuve d'Ascq,France (Christine Faille, Christine.faille@lille.inra.fr).

A growth medium was prepared as follows: powder meat extracts (Biokar)were dissolved in 0.1% distilled water and sterilized. 20 ml of inoculumwith 5.10⁷ CFU/ml in a solution of Trypticase Soy Broth (TSB, Biokar)were deposited on the surface of the cylinders. These cylinders wereincubated in a moist chamber at 30° C. for 2 hours to allow adhesion ofthe cells. The TSB solution was then removed and replaced by 20 ml ofthe meat growth medium and the cylinders were incubated 24 h at 30° C.The growth medium was then replaced by fresh meat medium and thecylinders were again incubated for 24 h.

Cleaning procedure comprised placing the cylinders contaminated with thebiofilm in straight pipes of a pilot industrial plant cleaned bycirculation (CIP cleaning). The treatment solutions were circulated for30 minutes at 45° C. at a flow rate of 300 l/h. For two of the strains,tests were also performed at a flow rate of 600 l/h. Each test wasrepeated three times and a mean was determined.

The development and removal of the biofilms were monitored by microscopeobservation and by quantification of viable cells on the cylinders via acount on Trypticase Soy Broth medium after detachment of the cells.

Description of the Protocol for Preparation of the Composition

The detergent component was prepared by mixing in a determined volume ofwater: a phosphonate, a polyacrylate and an anionic wetting agent. Therespective proportions in the detergent component were 3%, 4% and 3%.The pH of the solution was brought to 13.3 by dilution. A solution ofthe enzymatic component was prepared. This comprised a proportion of 30%proteases (EC 3.4.21), 30% laccase (EC 1.10.3.2) and 10% alpha-amylase(EC 3.2.1.1) and a conventional excipient.

The pH of the solution of the enzymatic component was brought to 9 byprogressively adding a solution of potassium hydroxide. The solution ofthe composition according to the invention was prepared by adding thedetergent component and enzymatic component to water. The solution ofthe said composition of the invention comprised 1% detergent componentand 0.05% enzymatic component. The pH of the solution of saidcomposition according to the invention was approximately 10.

Description of the Tests and Test Results

Test with the Pseudomonas fluorescens Strain

The graph given in FIG. 1 shows the quantity of biofilm present on thewall of the cylinder after application of treatment solutions followingthe conditions set forth above. The surfaces of the substrate werecoated with a biofilm formed by the Pseudomonas fluorescens strain. Theinitial quantity of biofilm present before treatment is denoted A1 inFIG. 1.

Three solutions were compared: a solution solely containing water (B1);0.5% sodium hydroxide solution (C1) and a solution of the composition ofthe invention (D1). The solutions were applied at a rate of 300 l/h andtemperature of 45° C. Solution (B1) comprising water allowed evaluationof flow-induced mechanical detachment of the biofilms.

It was observed that the biofilm resisted mechanical detachment sincethe quantities of biofilm are identical (curves A1 and B1). Theapplication of a solution of the composition of the invention allowed amajor reduction in the biofilm (curve D1, 103 CFU) compared with asodium hydroxide solution (curve C1, 10 ⁵ CFU). The composition of theinvention is therefore more efficient than a reference treatment (sodiumhydroxide solution) on a biofilm resistant to mechanical detachment.

FIG. 4 shows several images of the surface of a substrate, taken underepifluorescence microscopy, after application of the different treatmentsolutions at 300 l/h. Image (B4) shows the surface of the substratesubjected to a water solution, image (C4) shows the surface of thesubstrate subjected to a 0.5% sodium hydroxide solution and image (D4)shows the surface of the substrate subjected to a solution of thecomposition of the invention.

These data clearly show that the composition of the invention removedthe entirety of the matrix of the biofilms (diffuse staining) leavingonly isolated cells on the surfaces or small groups of cells distributedover a single layer unprotected by a matrix. The sodium hydroxidesolution on the other hand did not allow complete removal of the matrix(diffuse staining) which still partly protects residual cells. Asubsequent disinfection phase will therefore be much more efficientafter application of a solution of the composition according to theinvention.

Test with the Bacillus mycoides Strain

The graph given in FIG. 2 shows the quantity of biofilm present on thewall of the cylinder after applications of treatment solutions followingthe conditions set forth above. The surfaces of the substrate werecoated with a biofilm formed by the Bacillus mycoides strain. The amountof biofilm present before treatment is shown by curve (A2).

Five solutions were compared: a solution solely containing water (B2),0.5% sodium hydroxide solution (C2), a solution of the compositionaccording to the invention (D2 and F2) and a 2% sodium hydroxidesolution (E2). The solutions A2-D2 were applied at a flow rate of 300l/h whilst the solutions E2-F2 were applied at a flow rate of 600 l/h.

The biofilm of Bacillus mycoides is relatively sensitive to mechanicaldetachment its quantity being reduced by 90% with the application ofsolution (B2). Application of the solution of the composition of theinvention (D2 and F2) showed equivalent performance to the sodiumhydroxide solution (C2 and E2) whether the flow rate was 300 l/h or 600l/h.

FIG. 5 shows several images of the surface of the substrate taken underepifluorescence microscopy after application of the different treatmentsolutions at 300 l/h. Image (BS) shows the surface of the substratesubjected to a water solution, image (C5) shows the surface of thesubstrate subjected to a 0.5% sodium hydroxide solution, and image (D5)shows the surface of the substrate subjected to a solution of thecomposition of the invention.

These clearly show that the composition of the invention removed most ofthe matrix of the biofilms, only leaving isolated cells or small groupsof cells distributed over a single layer unprotected by a matrix. Thesodium hydroxide solution did not allow more efficient removal of thebiofilm. The subsequent action of a disinfectant solution will thereforebe more efficient if a solution of the composition of the invention ispreviously applied.

Test with the Bacillus cereus Strain

The graph give in FIG. 3 shows the amount of biofilm present on the wallof the cylinder after application of treatment solutions following theconditions set forth above. The surfaces of the substrate were coatedwith a biofilm formed by the Bacillus cereus strain. The amount ofbiofilm present before treatment is shown by curve A3.

Five solutions were compared: a solution solely containing water (B3),0.5% sodium hydroxide solution (C3), a solution of the composition ofthe invention (D3 and F3) and a 2% sodium hydroxide solution (E3).Solutions B3, C3, D3 were applied at a flow rate of 300 l/h, whilstsolutions E3 and F3 were applied at a faster rate (600 l/h). A fasterflow rate has an influence on mechanical detachment.

The biofilm of Bacillus cereus fully resists mechanical detachment(curve A3 and B3). The composition of the invention showed greaterefficacy at a flow rate of 300 l/h compared with a 0.5% sodium hydroxidesolution (curve C3) and even at a flow rate of 600 l/h compared with amore concentrated sodium hydroxide solution (curve E3).

FIG. 6 shows several images of the surface of a substrate, taken underepifluorescence microscopy, after application of the different treatmentsolutions. Image (B6) shows the surface of the substrate subjected to asolution of water, image (C6) shows the surface of the substratesubjected to a 0.5% sodium hydroxide solution and image (D6) shows thesurface of the substrate subjected to a solution of the compositionaccording to the invention.

These images again show that the composition of the invention removesalmost all the matrix of the biofilms, only leaving isolated cells orsmall groups of cells distributed over a single layer unprotected by amatrix.

The sodium hydroxide solution does not allow efficient removal of thebiofilm. The disinfection phase of the substrate after the removal phaseof the biofilm will therefore be more efficient after application of asolution of the composition of the invention for this removal phase.

The different tests performed in this Example demonstrate the efficacyof the composition of the invention in removing a biofilm from asubstrate. The composition of the invention proves to be more efficientthan a sodium hydroxide solution routinely used in the prior art. Thecomposition of the invention also exhibits high efficacy against severaltypes of biofilm derived from different bacteria. The composition of theinvention therefore brings an efficient solution to the problems raisedin the prior art and known to those skilled in the art.

Example 2

Tests were also performed on two industrial production lines (butter andmargarine production). Stainless steel test coupons (8*1 cm) were placedfor 15 days in production circuits having sporadic contamination. Thesecoupons were therefore subjected to cycles of production and standardcleaning of the installations.

The presence of biofilm in the plant was determined by the developmentof a biofilm on the coupons. Specific Clean-In-Place procedure using thecomposition of the invention was applied in the plant in the presence ofthe contaminated coupons, and whose efficacy was demonstrated throughthe removal of the biofilms formed on the coupons.

The presence and size of the biofilms on the coupons were determined ina laboratory by staining the coupons and comparing the staining obtainedwith a visual scale (HYDROBIO®, BKG) and by observation under an opticalmicroscope (40× and 100×).

Two cleaning protocols were tested: the first protocol was used asreference and corresponded to the application of solutions known in theprior art, the second protocol included the addition of a step to applya solution of the composition according to the invention.

In particular Protocol I comprised the following steps: cleaning withwater, alkaline cleaning, rinsing, sanitization (or disinfection) andfinal rinsing. Protocol I was applied every 32 hours for one week.Cleaning with water was applied for 15 minutes at a temperature ofbetween 60 and 65° C. Alkaline cleaning allows pipes to be cleaned oforganic matter present after a production cycle. This step was conductedusing a 3.5% sodium hydroxide solution applied for 2*15 minutes at atemperature of between 70-75° C.

The sanitization step allows the removal of germs which may still bepresent but are unprotected by the matrix of the biofilm. This stepentailed the application of a 1.5% solution of Deptil OX (HYPRED) for2*15 minutes at a temperature of between 20 and 25° C. The Deptil OXsolution contains a mixture of peracetic acid and hydrogen peroxide.

The rinsing steps were performed for 15 minutes at a temperature ofbetween 20 and 25° C.

Protocol II differed from Protocol I in that it comprised a specificcleaning step before the sanitization (or disinfection) step. Inaddition, Protocol II was only applied once.

The specific cleaning step consisted of the application of a solution ofthe composition according to the invention. This mixture was applied for30 minutes at a temperature of between 40 and 45° C. The resultsobtained are given in Table 1.

TABLE 1 Quantity of biofilm after Quantity of biofilm after Protocol I(g/m²) Protocol II (g/m²) Production line 1 25 <5 Production line 2 25to 35 <5

Protocol I did not allow the limiting and prevention of growth of abiofilm despite the sanitization (disinfection) step. The biofilm whichhad formed on the stainless steel coupons at the heart of theinstallation was therefore very resistant. The application of ProtocolII including a specific cleaning step with a solution of the compositionof the invention allowed much more efficient removal of this biofilm(below the detection limit). Greater efficacy of the specific cleaningwas therefore demonstrated which therefore provides an innovativesolution to prior art problems.

Example 3

Tests allowing a comparison between optimal pH values were conducted.

Biofilms were developed on stainless steel coupons previously washed andsterilized. An inoculum of bacterium (Pseudomonas aeruginosa) wasprepared by culture for 16 h in a 10% TSB medium (Tryptone Soy Broth).This pre-culture was diluted to obtain an optical density (OD) of 0.05to 600 nm, and 500 μl of this solution were spread on each coupon. Thecoupons were incubated 48 h in Petri dishes held under moist conditionsand placed in an oven at 30° C. After 2 hours, the bacterial solutionwas replaced by 500 μl of fresh 10% TSB medium. After 24 h, this culturemedium was renewed.

The coupons were then cleaned and placed on metal rods separated bynuts. They were then placed in the cleaning solutions. The coupons wereleft immersed in the above-mentioned cleaning solutions for a total timeof 30 minutes.

The cleaning solutions were the following:

-   -   1. Composition of the invention at pH 4.5, immersion for 30        minutes    -   2. Composition of the invention at pH7, immersion for 30 minutes    -   3. Composition of the invention at pH 9.7, immersion for 30        minutes

The coupons were subsequently placed in test tubes containing 10 ml ofTSB solution+0.5% Tween 80 for bacterial count The test tubes wereincubated for 5 minutes on the table before being sonicated for 2 and ahalf minutes and vortexed for 30 seconds. The incubation, sonication andvortex steps were repeated.

The coupons were afterwards collected and the medium comprising the TSBsolution+0.5% Tween was subjected to serial dilutions using peptonewater (peptone water free of indole: preparation of 10 ml of 15 g/ldissolution. Dilution of 1 ml of this solution in 1 litre of sterilewater); this medium was then spread on Petri dishes and incubated at 30°C. overnight before counting.

The biofilm was also stained as follows: the test coupons were drainedand immersed in the staining solution for 10 minutes, selective for theproteins of the biofilm matrix. The coupons were then immersed in thedifferent above-mentioned cleaning solutions 1 to 3, for a time of twice10 minutes (the cleaning solutions were replaced between the twocleaning steps) before being left to dry in open air.

Results

At acid pH (4.5): a mat of bacteria developed on the surface of thePetri dishes, which indicated the presence of a large quantity ofresidual biofilm on the coupons.

At neutral pH (7): a mat of bacteria developed on the surface of thePetri dishes.

At alkaline pH (9.7): no bacteria present in the Petri dishes.

FIG. 7 gives the results of the staining of the biofilms. FIG. 7Aillustrates the references before cleaning with treatment solutions 1 to3, whilst FIG. 7B illustrates the steel coupons after cleaning withsolutions 1 to 3 from left to right. As can be seen, cleaning atalkaline pH with the composition of the invention is clearly moreefficient than at acid pH or at neutral pH, the biofilm developed on thecoupon being fully detached with alkaline pH whereas it persists atother pH values.

Comparative Example

A test intended to determine the efficacy of four enzymes against threebacterial strains was conducted.

The results of this test are given in FIGS. 8 to 10. Biofilms wereformed on steel coupons (12) as in Example 3.

4 coupons were therefore produced for each bacterial strain (Pseudomonasfluorescens, Bacillus cereus and Chryseobacterium meningosepticum). Thecoupons were then rinsed with water before each being incubated with acleaning solution respectively containing a first, a second protease, alaccase and a polysaccharidase for 30 minutes.

Tables 2 gives the results of the bacterial counts whilst FIGS. 8 to 10show the logarithm of the results of bacterial count along the Y-axisand the type of enzyme used along the X-axis.

TABLE 2 Chryseobacterium Pseudomonas meningosepticum Bacillus cereusfluorescens Initial contamination 2.0 × 10⁸ 7.0 × 10⁶ 2.0 × 10⁸ levelFirst protease 4.0 × 10⁵ 7.0 · 10² 1.0 × 10² Second protease 2.0 × 10⁶9.0 × 10³ 3.8 × 10⁴ Laccase 8.0 × 10³ 1.3 × 10⁵ 1.6 × 10² Amylase 1.0 ×10⁵ 9.6 × 10⁴ 3.9 × 10⁶

As can be seen, some bacterial strains are more sensitive to the actionof the polysaccharidase and laccase, as is the case for Chryseobacteriummeningosepticum (Gram-). As for Bacillus Cereus (Gram+), this is moresensitive to the combination of the two proteases whereas Pseudomonasfluorescens (Gram−) is more sensitive to the combination of the firstprotease and the laccase.

Evidently, the present invention is in no way limited to theabove-described embodiments and numerous modifications can be madethereto without departing from the scope of the appended claims. Forexample, the fields of application of the compositions of the inventionhave proved to have particular use for cleaning by immersion e.g. in thehospital sector (cleaning laboratory equipment, surgical equipment), orby circulation within piping e.g. in the agri-food industry, in airconditioning installations, in water purifying and desalinating plants,for sailing vessel hulls and any type of immersed equipment, in washingmachine, dishwasher circuits etc., or for soaking and circulation inpiping.

1. A method for removing a biofilm present on a substrate, the methodcomprising the steps of: (a) providing a detergent component comprisinga sequestrant, a wetting agent and a dispersant, and providing anenzymatic component containing at least one protease, and at least onepolysaccharidase; (b) placing the detergent component in solution in anaqueous phase; (c) placing the enzymatic component in solution in thesolution formed at step (b) to form a solution comprising thesequestrant, the wetting agent, the dispersant, the at least oneprotease, and the at least one polysaccharidase; or (b′) placing theenzymatic component in solution in an aqueous phase; (c′) placing thedetergent component in solution in the solution formed at step (b′) toform a solution comprising the sequestrant, the wetting agent, thedispersant, the at least one protease, and the at least onepolysaccharidase, or optionally steps (b) and (c), or steps (b′) and(c′), are conducted simultaneously to provide a solution comprising thesequestrant, the wetting agent, the dispersant, the protease, and thepolysaccharidase; (d) applying the solution formed at step (c) or (c′)to the substrate for a predetermined period of time to effect biofilmremoval from the substrate, wherein the said sequestrant is selectedfrom the group consisting of a phosphonate, a phosphinate, a phosphate,glucono-delta-lactone, sodium gluconate, potassium gluconate, calciumgluconate, a citrate, a salt thereof, and a mixture thereof, whereinsaid wetting agent is non-foaming.
 2. The method according to claim 1,wherein biofilm removal from the substrate removes microorganisms fromthe substrate.
 3. The method according to claim 1 further comprisingapplying a biocide to the substrate.
 4. The method according to claim 1,wherein applying the solution to the substrate at step (d) comprisesapplying the solution to surface of a food production line.
 5. Themethod according to claim 1, wherein applying the solution to thesubstrate at step (d) comprises circulating the solution within piping.6. The method according to claim 1, wherein the substrate is a floor orsurface.
 7. The method according to claim 1, wherein the method is aclean-in-place method.
 8. The method according to claim 1, wherein themethod is an immersion method.
 9. The method according to claim 1,wherein the biofilm is a food industry biofilm and the substrate is afloor or surface in a food production line.
 10. The method according toclaim 1, wherein the predetermined period of time is between 15 minutesand 4 hours, preferably between approximately 30 to
 50. 11. The methodaccording to claim 1, wherein at least one enzymatic component of thesolution formed at step (c) or (c′) comprises a proportion ofprotease(s) the at least one protease of between 10 and 50%, and aproportion of polysaccharidase(s) the at least one polysaccharidase ofbetween 5 and 20% by weight relative to the total weight of theenzymatic component.
 12. The method according to claim 1, wherein thesolution formed at step (c) or (c′) comprises about 1% by weightdetergent component relative to the total weight of the solution andabout 0.05% by weight enzymatic component relative to the total weightof the solution.
 13. The method according to claim 1, wherein said atleast one polysaccharidase is an alpha-amylase.
 14. The method accordingto claim 1, wherein said at least one detergent component comprises aproportion of sequestrant of between 1 and 10% by weight relative to thetotal weight of the detergent component.
 15. The method according toclaim 1, wherein said at least one detergent component comprises aproportion of dispersant of between 1 and 10% by weight relative to thetotal weight of the detergent component.
 16. The method according toclaim 1, wherein said at least one detergent component comprises aproportion of wetting agent of between 1 and 15% by weight relative tothe total weight of the detergent component.
 17. The method according toclaim 1, wherein said dispersant is a polymer soluble or partly solublein water and is selected from the group consisting of polyethyleneglycol, cellulose derivatives, a polymer comprising at least one acrylicacid or acrylic ester repeat unit, a polymer comprising at least oneacrylic acid or acrylic ester repeat unit of general formula—(CH₂—CH—COOR)— where R represents a hydrogen, alkyl or substitutedalkyl, aryl or substituted aryl group, a polymer of acrylic acid, and ahomopolymer of acrylic acid having a weight average molecular weightapproximately of between 2000 and
 6000. 18. The method according toclaim 1, wherein the dispersant is a polymer comprising at least oneacrylic acid or acrylic ester repeat unit.
 19. The method according toclaim 1, wherein said dispersant is a polymer having a weight averagemolecular weight of between approximately 500 and
 10000. 20. The methodaccording to claim 1, wherein said wetting agent is non-foaming wettingagent when hot.
 21. The method according to claim 1, wherein saidwetting agent is selected from the group consisting of C₆ to C₁₀ sodiumalkyl sulphates, C₆ to C₁₀ alcohol ether sulphates, C₆ to C₁₀ alkyl arylsulphonates and sucrose esters.
 22. The method according to claim 1,wherein the enzymatic component is in the form of a dry solid, a powder,or a lyophilizate.
 23. The method according to claim 1, wherein theenzymatic component further comprises a laccase.